Method for Evaluating and Comparing Immunorepertoires

ABSTRACT

Disclosed is a method for amplifying RNA and/or DNA from immune cell populations and using the amplified products to produce an immune response profile and evaluate the possible correlation between a normal or abnormal immune response and the development of a disease such as an autoimmune disease, cancer, diabetes, or heart disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. provisionalpatent application No. 61/045,586, filed Apr. 16, 2008.

FIELD OF THE INVENTION

The invention relates to methods for identifying biomarkers and tomethods for identifying T-cell receptor, antibody, and MHCrearrangements in a population of cells.

BACKGROUND OF THE INVENTION

Scientists have known for a number of years that certain diseases areassociated with particular genes or genetic mutations. Geneticcausation, however, accounts for only a portion of the diseasesdiagnosed in humans. Many diseases appear to be linked in some way tothe immune system's response to infectious and environmental agents, buthow the immune system plays a role in diseases such as cancer,Alzheimer's, costochondritis, fibromyalgia, lupus, and other diseases isstill being determined.

The human genome comprises a total number of 567-588 Ig (immunoglobulin)and TR (T cell receptor) genes (339-354 Ig and 228-234 TR) per haploidgenome, localized in the 7 major loci. They comprise 405-418 V, 32 D,105-109 J and 25-29 C genes. The number of functional Ig and TR genes is321-353 per haploid genome. They comprise 187-216 V, 28 D, 86-88 J and20-21 C genes (http://imgt.cines.fr). Through rearrangement of thesegenes, it has been estimated that approximately 2.5×10⁷ possibleantibodies or T cell receptors can be generated.

Although, at the germine level, human beings are capable of generatinglarge numbers of diverse Igs and TRs, the number of available Igs andTRs for a particular individual is actually much smaller due to negativeselection during B and T cell development. In some individuals, thisprocess may not remove some of the cells that would cross-react with thebody's own tissues, and this may be the cause of some types ofautoimmune diseases.

A few diseases to date have been associated with the body's reaction toa common antigen (Prinz, J. et al., Eur. J. Immunol. (1999) 29(10):3360-3368, “Selection of Conserved TCR VDJ Rearrangements in ChronicPsoriatic Plaques Indicates a Common Antigen in Psoriasis Vulgaris”)and/or to specific VDJ rearrangements (Tamaru, J. et al., Blood (1994)84(3): 708-715, “Hodgkin's Disease with a B-cell Phenotype Often Shows aVDJ Rearrangement and Somatic Mutations in the V_(H) Genes”). What isneeded is a better method for evaluating changes in human immuneresponse cells and associating those changes with specific diseases.

SUMMARY OF THE INVENTION

The invention relates to a method for producing an immune status profile(ISP) for a human and/or animal. In one aspect of the invention, themethod comprises the steps of amplifying, in a first amplificationreaction using target-specific primers, at least one RNA and/or DNA froma sample of white blood cells from at least one human or animal subjectto produce at least one amplicon, at least a portion of thetarget-specific primers comprising additional nucleotides to incorporateinto a resulting amplicon a binding site for a common primer; rescuingthe at least one amplicon from the first amplification reaction;amplifying, by the addition of common primers in a second amplificationreaction, the amplicons of the first amplification reaction having atleast one binding site for a common primer; and sequencing the ampliconsof the second amplification reaction to identify and quantify DNAsequences representing antibody and/or receptor rearrangements to createan immune status profile.

In another aspect of the invention, the step of rescuing the at leastone amplicon from the first amplification reaction may be omitted, andthe first and second amplification reactions may occur withoutseparation of the amplicons from the target-specific primers. GenomicDNA may also be amplified, and the step of amplifying DNA may besubstituted for the step of amplifying RNA, especially in cases whereanalysis of an immune system component such as the majorhistocompatibility complex (MHC) is desired.

In aspects of the invention, subpopulations of white blood cells may beisolated by flow cytometry to separate naïve B cells, mature B cells,memory B cells, naïve T cells, mature T cells, and memory T cells. Invarious aspects of the method, recombinations in the subpopulation ofcells are rearrangements of B-cell immunoglobulin heavy chain (IgH),kappa and/or lambda light chains (IgK, IgL), T-cell receptor Beta,Gamma, Delta, and/or Major Histocompatibility Complex (MHC) molecules Ior II.

In another aspect of the invention, the method may also comprisecompiling and comparing the immune cell profile for a population ofnormal individuals with the immune cell profile for a population ofindividuals who have been diagnosed with a disease to determine if thereis a correlation between a specific rearrangement or set ofrearrangements and the disease.

In another aspect of the invention, the method may comprise comparingthe immune cell profile identified for a population of individuals towhom a vaccine has been administered with the immune cell profile for apopulation of individuals to whom the vaccine was not administered toevaluate the efficacy of the vaccine in producing an immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a and FIG. 1 b are photographs of gels illustrating the presenceof amplification products obtained by the method of the invention usingprimers disclosed herein.

FIG. 2 illustrates distributions of domain usage for (a) Ig heavy chainin healthy control sample, (b) TCR beta chain in a blood sample from apatient with colon cancer, (c) Ig kappa chain in a blood sample from apatient with Chronic Lymphocytic Leukemia (CLL), and (d) Ig lambda chainin a blood sample from a patient with Systemic lupus erythematosus(SLE). Empty spots indicate missing sequences associated withcorresponding V-J combinations and the height of the column indicatesthe frequency of occurrence of a particular sequence.

DETAILED DESCRIPTION

The inventor has developed a method for evaluating antibody and receptorrearrangements from a large number of cells, the method being useful forcomparing rearrangements identified in populations of individuals todetermine whether there is a correlation between a specificrearrangement or set of rearrangements and a disease, or certainsymptoms of a disease. The method is also useful for establishing ahistory of the immune response of an individual or individuals inresponse to infectious and/or environmental agents, as well as forevaluating the efficacy of vaccines.

The invention relates to a method for producing an immune status profile(ISP) for a human and/or animal. In one aspect of the invention, themethod comprises the steps of amplifying, in a first amplificationreaction using target-specific primers, at least one RNA from a sampleof white blood cells from at least one human or animal subject toproduce at least one amplicon, at least a portion of the target-specificprimers comprising additional nucleotides to incorporate into aresulting amplicon a binding site for a common primer; rescuing the atleast one amplicon from the first amplification reaction; amplifying, bythe addition of common primers in a second amplification reaction, theamplicons of the first amplification reaction having at least onebinding site for a common primer; and sequencing the amplicons of thesecond amplification reaction to identify and quantify DNA sequencesrepresenting antibody and/or receptor rearrangements to create an immunestatus profile.

Where the term “comprising” is used herein, “consisting essentially of”and “consisting of” may also be used. The term “immune status profile”is intended to mean a profile for an individual or population ofindividuals indicating the presence and/or absence of sequencesrepresenting specific rearrangements representing the diversity of Bcells, T cells, and/or other cells of the human and/or animal immunesystem, as well as the frequency of their occurrence. Where ampliconsare referred to as “rescued” herein, it is to be understood thatamplicon rescue may occur by the separation of amplicons from theprimers which are used to create them, or may occur by dilution of theamplicon/primer mix so that, by virtue of the fact that there aresignificantly more amplicons than primers from a first amplificationreaction, the effect of those primers is minimized in a secondamplification reaction using different primers. “Common primers” arethose primers that may be used to amplify polynucleotides (e.g.,amplicons from a first amplification produced by target-specificprimers) having non-identical sequences in general, but sharing sequencesimilarities in that they contain binding sites for the same primers.Common primers are generally chosen for their efficiency at primingsuccessful amplifications, so their use is effective for achievinghigher levels of amplification in a non-target-specific manner in themethod of the present invention. Common primer binding sites may beincorporated into amplicons resulting from a first amplification byattaching their sequences or their complementary sequences to thesequence of a target-specific primer. Common primers may be chosen byone of skill in the art by a variety of primer-design methods.

Subpopulations of white blood cells may be isolated by flow cytometry toseparate naïve B cells, mature B cells, memory B cells, naïve T cells,mature T cells, and memory T cells. Recombinations in thesesubpopulations of cells are generally rearrangements of B-cellimmunoglobulin heavy chain (IgH), kappa and/or lambda light chains (IgK,IgL), T-cell receptor Beta, Gamma, Delta, and/or MajorHistocompatibility Complex (MHC) molecules I or II.

By performing an additional step, namely that of compiling and comparingthe average immune status profile for a population of normal individualswith an average immune status profile for a population of individualswho have been diagnosed with a disease, it is possible to use the immunecell profile to determine if there is a correlation between a specificrearrangement or set of rearrangements and the disease.

The invention also provides a method for evaluating vaccine efficacy, interms of creating a change in the immune cell profile, by performing thesteps of the method and comparing the immune cell profile identified fora population of individuals to whom a vaccine has been administered withthe immune cell profile for a population of individuals to whom thevaccine was not administered to evaluate the efficacy of the vaccine inproducing an immune response.

In one embodiment of the invention, a peripheral blood sample is takenfrom a patient and isolation of a subpopulation of white blood cells maybe performed by flow cytometry to separate naïve B cells, mature Bcells, memory B cells, naïve T cells, mature T cells, and memory Tcells. In various embodiments of the method, recombinations in thesubpopulation of cells may comprise rearrangements of B-cellimmunoglobulin heavy chain (IgH), kappa and/or lambda light chains (IgK,IgL), T-cell receptor Beta, Gamma, Delta, or Major HistocompatibilityComplex (MHC) molecules I or II.

In some aspects, the step of rescuing the amplicons from the firstamplification reaction may be omitted and the two amplificationreactions may be performed in the same reaction tube without ampliconrescue or dilution of the primers remaining from the first amplificationreaction.

The inventor previously developed a PCR method known as tem-PCR, whichhas been described in publication number WO2005/038039. More recently,the inventor has developed a method called amplicon rescue multiplexpolymerase chain reaction (arm-PCR), which is described in U.S.PCT/US09/39552 and herein. Both the tem-PCR and arm-PCR methods providesemi-quantitative amplification of multiple polynucleotides in onereaction. Additionally, arm-PCR provides added sensitivity. Both providethe ability to amplify multiple polynucleotides in one reaction, whichis beneficial in the present method because the repertoire of various Tand B cells, for example, is so large. The addition of a common primerbinding site in the amplification reaction, and the subsequentamplification of target molecules using common primers, gives aquantitative, or semi-quantitative result—making it possible todetermine the relative amounts of the cells comprising variousrearrangements within a patient blood sample. Clonal expansion due torecognition of antigen results in a larger population of cells whichrecognize that antigen, and evaluating cells by their relative numbersprovides a method for determining whether an antigen exposure hasinfluenced expansion of antibody-producing B cells or receptor-bearing Tcells. This is helpful for evaluating whether there may be a particularpopulation of cells that is prevalent in individuals who have beendiagnosed with a particular disease, for example, and may be especiallyhelpful in evaluating whether or not a vaccine has achieved the desiredimmune response in individuals to whom the vaccine has been given.

There are several commercially available high throughput sequencingtechnologies, such as Roche Life Sciences 454 Sequencing®. In thissequencing method, 454A and 454B primers are either linked onto PCRproducts during PCR or ligated on after the PCR reaction. When done inconjunction with tem-PCR or arm-PCR, 454A and 454B primers may be usedas common primers in the amplification reactions. PCR products, usuallya mixture of different sequences, are diluted to about 200 copies perμl. In an “emulsion PCR” reaction, (a semisolid gel like environment)the diluted PCR products are amplified by primers (454A or 454B) on thesurface of the microbeads. Because the PCR templates are so dilute,usually only one bead is adjacent to one template, and confined in thesemisolid environment, amplification only occurs on and around thebeads. The beads are then eluted and put onto a plate with speciallydesigned wells. Each well can only hold one bead. Reagents are thenadded into the wells to carry out pyrosequencing. A fiber-optic detectormay be used to read the sequencing reaction from each well and the datais collected in parallel by a computer. One such high throughputreaction could generate up to 60 million reads (60 million beads) andeach read can generate about 300 bp sequences.

One aspect of the invention involves the development of a database ofimmune status profiles, or “personal immunorepertoires” (PIRs), so thateach individual may establish a baseline and follow the development ofimmune responses to antigens, both known and unknown, over a period ofyears. This information may, if information is gathered from a largenumber of individuals, provide an epidemiological database that willproduce valuable information, particularly in regard to the developmentof those diseases such as cancer and heart disease which are thought tooften arise from exposure to viral or other infectious agents, many ofwhich have as yet been unidentified. One particularly important use forthe method of the invention enables studies of children to determinewhether infectious disease, environmental agents, or vaccines may be thecause of autism. For example, many have postulated that vaccineadministration may trigger the development of autism. However, many alsoattribute that potential correlation to the use of agents such asthimerosol in the vaccine, and studies have demonstrated that thimerosoldoes not appear to be a causative agent of the disease. There is stillspeculation that the development of cocktail vaccines has correlatedwith the rise in the number of cases of autism, however, but gatheringdata to evaluate a potential causal connection for multiple antigens isextremely difficult. The method of the present invention simplifies thatprocess and may provide key information for a better understanding ofautism and other diseases in which the immune response of differentindividuals may provide an explanation for the differential developmentof disease in some individuals exposed to an agent or a group of agents,while others similarly exposed do not develop the disease.

Imbalances of the PIR, triggered by infection, may lead to manydiseases, including cancers, leukemia, neuronal diseases (Alzheimer's,Multiple Sclerosis, Parkinson's, autism etc), autoimmune diseases, andmetabolic diseases. These diseases may be called PIR diseases. There maybe two PIR disease forms. (1) a “loss of function” form, and (2) a “gainof function” form. In the “loss of function” form, a person issusceptible to a disease because his/her restricted and/or limited PIRlacks the cells that produce the most efficient and necessary Igs andTRs. In the “gain of function” form, a person is susceptible to adisease because his/her PIR gained cells that produce Igs and TRs thatnormally should not be there. In the “loss of function” (LOF) PIRdiseases, an individual does not have the appropriate functional B or Tcells to fight a disease. His/her HLA typing determines that those cellsare eliminated during the early stages of the immune cell maturationprocess, the cells generally being eliminated because they react tostrongly to his/her own proteins.

One aspect of the invention also comprises entering a patient immunecell profile into a database in combination with identifying informationsuch as, for example, a patient identification number, a code comprisingthe patient's HLA type, a disease code comprising one or more clinicaldiagnoses that may have been made, a “staging code” comprising the dateof the sample, a cell type code comprising the type of cellsubpopulation from which the RNA was amplified and sequenced, and one ormore sequence codes comprising the sequences identified for the sample.

The described method includes a novel primer set that not only allowsamplification of the entire immunorepertoire, but also allows multiplexamplification that is semi-quantitative. Multiplex amplificationrequires that only a few PCR or RT-PCR reactions are needed. Forexample, all immunoglobulin (Ig) sequences present may be amplified inone reaction, or two or three reactions may be performed separately,using primers specific for IgH, IgL or IgK. Similarly, the T-cellreceptors (TRs) may be amplified in just one reaction, or may beamplified in a few reactions including the T-cell receptors designatedTRA, TRB, TRD, and TRG. MHC genes may be amplified in just one PCRreaction. Semi-quantitative amplification allows all the targets in themultiplex reaction to be amplified independently, so that the end pointanalysis of the amplified products will reflect the original internalratio among the targets. Because this ratio is maintained, it ispossible to produce an immune cell profile that indicates the presenceor absence, as well as relative numbers, of various immune system cells.Amplification of RNA according to the method of the invention may beperformed using any or all of the primers listed in Tables 1, 2, and/or3. The invention therefore provides a method for using at least one(which may, of course, more preferably include a least 2, at least 3, atleast 4, etc.) primers chosen from among the group consisting of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ IDNO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ IDNO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68,SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ IDNO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87,SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 101, SEQ ID NO:102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO:129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ IDNO:138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 151, SEQ ID NO: 152,SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ IDNO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161,SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ IDNO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170,SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ IDNO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179,SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ IDNO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188,SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ IDNO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197,SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ IDNO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206,SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ IDNO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215,SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ IDNO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224,SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ IDNO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233,SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ IDNO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242,SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ IDNO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251,SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ IDNO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260,SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ IDNO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269,SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ IDNO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278,SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ IDNO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287,SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ IDNO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296,SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ IDNO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305,SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ IDNO: 310, SEQ ID NO: 311, SEQ ID NO: 312, and combinations thereof, toperform a first amplification producing at least one first amplicon, atleast one of the target-specific primers containing additional basepairs so that the amplification results in the addition of a bindingsequence for at least one common primer into the at least one firstamplicon; amplifying the at least one first amplicon in a secondamplification using at least one common primer to produce at least onesecond amplicon; and sequencing the at least one second amplicon toidentify and quantify the sequences produced by the first and secondamplifications. The listed primers were designed by the inventor toprovide efficient amplification of their respective RNA and/or DNAtargets. Use of the entire group of primers is effective to produce adetailed immune status profile for an individual. Use of a subset may,however, be desired when specific populations of T or B cells, forexample, are the subject of particular interest.

By way of further explanation, the following example may be illustrativeof the methods of the invention. Blood samples may be taken fromchildren prior to administration of any vaccines, those blood samplesfor each child being used in the method of the invention to create a“baseline” immune status profile or personal immunorepertoire (PIR) fromwhich future immune cell profiles, created from blood samples takenduring later years and analyzed by the method of the invention, may becompared. For each child, the future samples may be utilized todetermine whether there has been an exposure to an agent which hasexpanded a population of cells known to be correlated with a disease,and this may serve as a “marker” for the risk of development of thedisease in the future. Individuals so identified may then be moreclosely monitored so that early detection is possible, and any availabletreatment options may be provided at an earlier stage in the diseaseprocess.

The method of the invention may be especially useful for identifyingcommonalities between individuals with autoimmune diseases, for example,and may provide epidemiological data that will better describe thecorrelation between infectious and environmental factors and diseasessuch as heart disease, atherosclerosis, diabetes, and cancer-providingbiomarkers that signal either the presence of a disease, or the tendencyto develop disease.

The method may also be useful for development of passive immunitytherapies. For example, following exposure to an infectious agent,certain antibody-producing B cells and/or T cells are expanded. Themethod of the invention enables the identification of protectiveantibodies, for example, and those antibodies may be utilized to providepassive immunity therapies in situations where such therapy is needed.

The method of the invention may also provide the ability to accomplishtargeted removal of cells with undesirable rearrangements, the methodproviding a means by which such cells rearrangements may be identified.

The inventor has identified and developed target-specific primers foruse in the method of the invention. T-cell-specific primers are shown inTable 1, antibody-specific primers are shown in Table 2, andHLA-specific primers are shown in Table 3. Therefore, the method maycomprise using any combination of primers of Table 1, Table 2, and/orTable 3 to amplify RNA and/or DNA from a blood sample, and moreparticularly to identify antibodies, T-cell receptors, and HLA moleculeswithin a population of cells. For example, an analysis of T-celldistribution might utilize all or a portion of the primers listed inTable 1 (SEQ ID NO: 1 through SEQ ID NO: 157). An analysis of Ig mightutilize all or a portion of the primers listed in Table 2 (SEQ ID NO:158 through SEQ ID NO: 225), and an analysis of HLA distribution mightutilize all or a portion of the primers listed in Table 3 (SEQ ID NO:159 through SEQ ID NO: 312).

In a tem-PCR reaction, nested gene-specific primers are designed toenrich the targets during initial PCR cycling. Later, universal “Super”primers are used to amplify all targets. Primers are designated as F_(o)(forward out), F_(i) (forward in), R_(i) (reverse in), R_(o) (reverseout), FS (forward super primer) and RS, (reverse super primer), withsuper primers being common to a variety of the molecules due to theaddition of a binding site for those primers at the end of atarget-specific primer. The gene-specific primers (F_(o), F_(i), R_(i),and R_(o)) are used at extremely low concentrations. Different primersare involved in the tem-PCR process at each of the three major stages.First, at the “enrichment” stage, low-concentration gene-specificprimers are given enough time to find the templates. For each intendedtarget, depending on which primers are used, four possible products maybe generated: F_(o)/R_(o), F_(i)/R_(o), F_(i)/R_(i), and F_(o)/R_(i).The enrichment stage is typically carried out for 10 cycles. In thesecond, or “tagging” stage, the annealing temperature is raised to 72°C., and only the long 40-nucleotide inside primers (Fi and Ri) willwork. After 10 cycles of this tagging stage, all PCR products are“tagged” with the universal super primer sequences. Then, at the third“amplification” stage, high-concentration super primers work efficientlyto amplify all targets and label the pCR products with biotin during theprocess. Specific probes may be covalently linked with Luminex®color-coated beads.

To amplify the genes coding for immunoglobulin superfamily molecules,the inventor designed nested primers based on sequence informationavailable in the public domain. For studying B and T cell VDJrearrangement, the inventor designed primers to amplify rearranged andexpressed RNAs. Generally, a pair of nested forward primers is designedfrom the V genes and a set of reverse nested primers are designed fromthe J or C genes. The average amplicon size is 250-350 bp. For the IgHVgenes, for example, there are 123 genes that can be classified into 7different families, and the present primers are designed to befamily-specific. However, if sequencing the amplified cDNA sequences,there are enough sequence diversities to allow further differentiationamong the genes within the same family. For the MHC gene locus, theintent is to amplify genomic DNA.

The invention may be further described by means of the followingnon-limiting examples.

EXAMPLES Amplification of T or B Cell Rearrangement Sites

All oligos were resuspended using 1×TE. All oligos except 454A and 454Bwere resuspended to a concentration of 100 pmol/uL. 454A and 454B wereresuspended to a concentration of 1000 pmol/uL 454A and 454B arefunctionally the same as the common primers described previously, thedifferent sequences were used for follow up high throughput sequencingprocedures.

Three different primer mixes were made. An Alpha Delta primer mixincluded 82 primers (all of TRAV-C+TRDV-C), a Beta Gamma primer mixincluded 79 primers (all of TRBVC and TRGV-C) and a B cell primer mixthat included a total of 70 primers. F_(o), F_(i), and R_(i) primerswere at a concentration of 1 pmol/μL. R_(o) primers were at aconcentration of 5 pmol/uL. 454A and 454B were at a concentration of 30pmol/μL.

Three different RNA samples were ordered from ALLCELLS(www.allcells.com). All samples were diluted down to a finalconcentration of 4 ng/uL. The samples used were: ALL-PB-MNC (from apatient with acute lymphoblastic leukemia), NPB-Pan T Cells (normal Tcells) and NPB-B Cells (normal B cells).

RT-PCR was performed using a Qiagen® One-Step RT-PCR kit. Each samplecontained the following:

10 μL of Qiagen® Buffer

2 μL of DNTP's

2 μl of Enzyme

23.5 μL of dH2O

10 μL of the appropriate primer mix

2.5 μL of the appropriate template (long of RNA total)

The samples were run using the following cycling conditions:

50° C. for 30 minutes

95° C. for 15 minutes

94° C. for 30 seconds

15 cycles of

55° C. for 1 minute

72° C. for 1 minute

94° C. for 15 seconds

6 cycles of

70° C. for 1 minute 30 seconds

94° C. for 15 seconds

30 cycles of

55° C. for 15 seconds

72° C. for 15 seconds

72° C. for 3 minutes

4° C. Hold

The order of samples placed in the gel shown in FIG. 1 a was: (1) Ladder(500 bp being the largest working down in steps of 20 bp, the middlebright band in FIG. 1 a is 200 bp); (2) α+δ primer mix with 10 ng Pan TCells Template; (3) β+γ primer mix with 10 ng Pan T Cells Template; (4)B Cell primer mix with 10 ng B Cells Template; (5) B Cell primer mixwith 10 ng ALL Cells Template; (6) α+δ primer mix with 10 ng ALL CellsTemplate; (7) β+γ primer mix with 10 ng ALL Cells Template; 8. α+δprimer mix blank; (9) β+γ primer mix blank; (10) B Cell primer mixblank; (11) Running buffer blank. These samples were run on a pre-castClearPAGE® SDS 10% gel using 1× ClearPAGE® DNA native running buffer.

The initial experiment showed that a smear is generated from PCRreactions where templates were included. The smears indicate differentsizes of PCR products were generated that represented a mixture ofdifferent VDJ rearrangements. There was some background amplificationfrom the B cell reaction. Further improvement on that primer mix cleanedup the reaction.

To determine whether the PCR products indeed include different VDJrearrangements, it was necessary to isolate and sequence the singleclones. Instead of using the routine cloning procedures, the inventorused a different strategy. PCR products generated from the Alpha Deltamix and the Beta Gamma mix (lanes 2 and 3 in FIG. 1 a) were diluted1:1000 and a 2 μl aliquot used as PCR template in the followingreaction. Then, instead of using a mixture of primers that targeting theentire repertoire, one pair of specific F_(i) and R_(i) primers wereused (5 pmol each) to amplify only one specific PCR product. Thefollowing cycling conditions were used to amplify the samples:

95° C. for 5 minutes

30 cycles of

94° C. for 30 seconds

72° C. for 1 minute

72° C. for 3 minutes

4° C. hold

A Qiagen PCR kit was used to amplify the products. The Master Mix usedfor the PCR contained the following: 5 μL 10×PCR Buffer, 1 μL dNTP, 0.25μL HotStartTaq Plus, and 39.75 μL H₂O. (For a mix for 12 reactions: 60μL 10×PCR Buffer, 12 μL dNTP, 3 μL HotStartTaq Plus, and 477 μL H₂O.)

The photograph of the gel in FIG. 1 b shows the PCR products of thefollowing reactions: (1) Ladder; (2) TRAV1F_(i)+TRACR_(i) with alphadelta Pan T PCR product; (3) TRAV2F_(i)+TRACR_(i) with alpha delta Pan TPCR product; (4) TRAV3F_(i)+TRACR_(i) with alpha delta Pan T PCRproduct; (5) TRAV4F_(i)+TRACR_(i) with alpha delta Pan T PCR product;(6) TRAV5F_(i)+TRACR_(i) with alpha delta Pan T PCR product; (7)TRAV1F_(i)+TRACR_(i) with alpha delta Pan T PCR product; (8)TRAV2F_(i)+TRACR_(i) with alpha delta Pan T PCR product; (9)TRAV3F_(i)+TRACR_(i) with alpha delta Pan T PCR product; (10)TRAV4F_(i)+TRACR_(i) with alpha delta Pan T PCR product; (11)TRAV5F_(i)+TRACR_(i) with alpha delta Pan T PCR product; (12) PCR Blank.Primers listed as F₁ are “forward inner” primers and primers listed asF_(o) are “forward outer” primers, with R_(i) and R_(o) indicating“reverse inner” and “reverse outer” primers, respectively.

As illustrated by FIG. 1 b, a single PCR product was generated from eachreaction. Different size bands were generated from different reactions.This PCR cloning approach is successful for two major reasons—(1) ThePCR templates used in this reaction were diluted PCR products (1:1000)of previous reactions that used primer mixes to amplify all possible VDJrearrangements (for example, a primer mix was used that included totalof 82 primers to amplify T cell receptor Alpha and Delta genes) and (2)Only one pair of PCR primers, targeting a specific V gene, are used ineach reaction during this “cloning” experiment. In every case, a singleclone was obtained, and a specific T cell receptor V gene that matchedthe F_(i) primer was identified.

Sequencing of Immune Cell RNA Using Primers of SEQ ID NO: 1-SEQ ID NO:312

Pan-T, pan-B, and neutrophil isolation was performed usingsuper-paramagnetic polystyrene beads coated with monoclonal antibodyspecific for certain cell types (Dynabeads®, Invitrogen Corp., Carlsbad,Calif.) following manufacturer's instructions. Anti-CD3 beads were usedto isolate pan-T cells, anti-CD19 beads for pan-B cells, and anti-CD15beads for neutrophils. Isolated cells were resuspended in 300 μlRNAProtect® (Qiagen) reagent and counted using a hemacytometer.

T cell subpopulations were isolated from a normal patient 48-year-oldAsian male. PMBCs were obtained from 40 ml of whole blood collected insodium heparin by density centrifugation over Ficoll Prep Plus Reagent.Pan-T cells were isolated from the mononuclear layer using a magneticbead isolation kit (Miltenyi Biotec, Auburn, Calif.), followingmanufacturer's instructions. Anti-CD4 and anti-CD25 beads were used toisolate regulatory T cells, anti-CD56 for NKT cells, anti-CD8 forcytotoxic T cells, and anti-CD4 and anti-CD294 for Th2 cells. Th1 cellswere isolated via negative selection. A separate 40 ml sample of wholeblood collected in sodium heparin was used to obtain naive, activated,and memory T cell subpopulations. Anti-CD45RA beads were used to isolatenaive T cells, anti-CD69 for activated T cells and anti-CD45RO beads formemory T cells. Isolated cells were re-suspended in 300 μl ofRNAProtect® reagent (Qiagen). Cells were counted using a hemacytometer.

DNA extraction from the isolated neutrophils was performed using aQIAmp® DNA mini kit (Qiagen) using the protocol provided by themanufacturer. RNA was extracted from T cell subsets using an Rneasy® kit(Qiagen) according to the protocol provided by the manufacturer. Theconcentrations of extracted DNA and RNA were measured using Nanodrop®technology (Nanodrop Technologies, Wilmington, Del.). Samples werestored at −80° C.

RT-PCR was performed according to the method of the invention usingnested PCR to amplify multiple targets and target-specific primers toincorporate a common primer binding sequence into the resultingamplicons in a first amplification reaction. Common primers were thenused in a second amplification reaction to exponentially amplify theamplicons rescued from the first amplification reaction while preservingthe relative ratios of each amplicon. PCR was performed using a One-StepRT-PCR kit (Qiagen). DNA amplification for HLA typing was similarlyperformed, but with a mulitplex PCR kit (Qiagen). Each amplicon mixturewas subjected to high-throughput sequencing with the Roche 454sequencing platform.

More than 1.6 million effective sequences were generated for one singleindividual (normal 48-year-old Asian male) by sampling differentsubpopulations of lymphocytes in peripheral blood at different timepoints. Additionally, 170,734 effective sequences were generated for thecolon cancer, CLL, SLE, and a second healthy patient (a 32-year-oldCaucasian male). The number of unique reads generated in this study wascompared to the number of unique reads existing in public databases inTable 1. The public sequence data set was compiled by searching Genbanknucleotide database with terms of ‘human[orgn] AND (immunoglobulin[titl]OR T-cell receptor[titl]) AND mRNA[titl]’. In addition, the annotatedIMGT/LIGM-DB (Brezinschek et al, 1995) cDNA sequences were gathered witha Python script. The two data sets were merged, and one copy was keptfor any redundant sequences.

Biased usage of V, and J gene segments in a healthy control, CLL, coloncancer, and SLE sample was analyzed. The bias of domain usage wasparticularly outstanding for TCR beta chain in the colon cancer sampleand SLE sample, while in the healthy control sample the domain usage isquite normal without significant bias to any particular domain. It wasevident that colon cancer and SLE profiles not only show clonalexpansion, but demonstrate the loss of overall diversity, as well.

The distribution of functional germline V, J gene segments seen in thepan-T and pan-B populations from normal patient indicated that 87.2% ofpotential combinations have sequences observed. Only IGHV3-d was notobserved in this investigation, while TRBV4-3, IGHV3-d, IGHV4-30-4 andIGHV4-31 and IGHL3-22 were observed in other samples with extremely lowfrequency. Previous research did not reveal any cDNA sequence datarelated to IGHV3-d, which suggests that IGHV3-d may be usedinfrequently. Some sequences were present in high (e.g. 1000) numbers,while others were present in significantly lower numbers. The inventorbelieves that higher numbers represent lymphocyte clonal expansions,reflecting the real immune responses in the subject. Studies of VH genedistribution in normal individuals have previously found the frequencyof usage in general to be similar to the germline complexity, while manyimmune responses show some level of bias in the usage of V, D and J genesegments.

TABLE 1 Sequence Primer Sequence ID Number TRAV1Fo5′-TGCACGTACCAGACATCTGG-3′ SEQ ID NO: 1 TRAV1Fi AGGTCGTTTTTCTTCATTCC SEQID NO: 2 TRAV2Fo TCTGTAATCACTCTGTGTCC SEQ ID NO: 3 TRAV2FiAGGGACGATACAACATGACC SEQ ID NO: 4 TRAV3Fo CTATTCAGTCTCTGGAAACC SEQ IDNO: 5 TRAV3Fi ATACATCACAGGGGATAACC SEQ ID NO: 6 TRAV4FoTGTAGCCACAACAACATTGC SEQ ID NO: 7 TRAV4Fi AAAGTTACAAACGAAGTGGC SEQ IDNO: 8 TRAV5Fo GCACTTACACAGACAGCTCC SEQ ID NO: 9 TRAV5FiTATGGACATGAAACAAGACC SEQ ID NO: 10 TRAV6Fo GCAACTATACAAACTATTCC SEQ IDNO: 11 TRAV6Fi GTTTTCTTGCTACTCATACG SEQ ID NO: 12 TRAV7FoTGCACGTACTCTGTCAGTCG SEQ ID NO: 13 TRAV7Fi GGATATGAGAAGCAGAAAGG SEQ IDNO: 14 TRAV8Fo AATCTCTTCTGGTATGTSCA SEQ ID NO: 15 TRAV8FiGGYTTTGAGGCTGAATTTA SEQ ID NO: 16 TRAV9Fo GTCCAATATCCTGGAGAAGG SEQ IDNO: 17 TRAV9Fi AACCACTTCTTTCCACTTGG SEQ ID NO: 18 TRAV10FoAATGCAATTATACAGTGAGC SEQ ID NO: 19 TRAV10Fi TGAGAACACAAAGTCGAACG SEQ IDNO: 20 TRAV11Fo TCTTAATTGTACTTATCAGG SEQ ID NO: 21 TRAV11FiTCAATCAAGCCAGAAGGAGC SEQ ID NO: 22 TRAV12Fo TCAGTGTTCCAGAGGGAGCC SEQ IDNO: 23 TRAV12Fi ATGGAAGGTTTACAGCACAG SEQ ID NO: 24 TRAV13FoACCCTGAGTGTCCAGGAGGG SEQ ID NO: 25 TRAV13Fi TTATAGACATTCGTTCAAAT SEQ IDNO: 26 TRAV14Fo TGGACTGCACATATGACACC SEQ ID NO: 27 TRAV14FiCAGCAAAATGCAACAGAAGG SEQ ID NO: 28 TRAV16Fo AGCTGAAGTGCAACTATTCC SEQ IDNO: 29 TRAV16Fi TCTAGAGAGAGCATCAAAGG SEQ ID NO: 30 TRAV17FoAATGCCACCATGAACTGCAG SEQ ID NO: 31 TRAV17Fi GAAAGAGAGAAACACAGTGG SEQ IDNO: 32 TRAV18Fo GCTCTGACATTAAACTGCAC SEQ ID NO: 33 TRAV18FiCAGGAGACGGACAGCAGAGG SEQ ID NO: 34 TRAV19Fo ATGTGACCTTGGACTGTGTG SEQ IDNO: 35 TRAV19Fi GAGCAAAATGAAATAAGTGG SEQ ID NO: 36 TRAV20FoACTGCAGTTACACAGTCAGC SEQ ID NO: 37 TRAV20Fi AGAAAGAAAGGCTAAAAGCC SEQ IDNO: 38 TRAV21Fo ACTGCAGTTTCACTGATAGC SEQ ID NO: 39 TRAV21FiCAAGTGGAAGACTTAATGCC SEQ ID NO: 40 TRAV22Fo GGGAGCCAATTCCACGCTGC SEQ IDNO: 41 TRAV22Fi ATGGAAGATTAAGCGCCACG SEQ ID NO: 42 TRAV23FoATTTCAATTATAAACTGTGC SEQ ID NO: 43 TRAV23Fi AAGGAAGATTCACAATCTCC SEQ IDNO: 44 TRAV24Fo GCACCAATTTCACCTGCAGC SEQ ID NO: 45 TRAV24FiAGGACGAATAAGTGCCACTC SEQ ID NO: 46 TRAV25Fo TCACCACGTACTGCAATTCC SEQ IDNO: 47 TRAV25Fi AGACTGACATTTCAGTTTGG SEQ ID NO: 48 TRAV26FoTCGACAGATTCMCTCCCAGG SEQ ID NO: 49 TRAV26Fi GTCCAGYACCTTGATCCTGC SEQ IDNO: 50 TRAV27Fo CCTCAAGTGTTTTTTCCAGC SEQ ID NO: 51 TRAV27FiGTGACAGTAGTTACGGGTGG SEQ ID NO: 52 TRAV29Fo CAGCATGTTTGATTATTTCC SEQ IDNO: 53 TRAV29Fi ATCTATAAGTTCCATTAAGG SEQ ID NO: 54 TRAV30FoCTCCAAGGCTTTATATTCTG SEQ ID NO: 55 TRAV30Fi ATGATATTACTGAAGGGTGG SEQ IDNO: 56 TRAV34Fo ACTGCACGTCATCAAAGACG SEQ ID NO: 57 TRAV34FiTTGATGATGCTACAGAAAGG SEQ ID NO: 58 TRAV35Fo TGAACTGCACTTCTTCAAGC SEQ IDNO: 59 TRAV35Fi CTTGATAGCCTTATATAAGG SEQ ID NO: 60 TRAV36FoTCAATTGCAGTTATGAAGTG SEQ ID NO: 61 TRAV36Fi TTTATGCTAACTTCAAGTGG SEQ IDNO: 62 TRAV38Fo GCACATATGACACCAGTGAG SEQ ID NO: 63 TRAV38FiTCGCCAAGAAGCTTATAAGC SEQ ID NO: 64 TRAV39Fo TCTACTGCAATTATTCAACC SEQ IDNO: 65 TRAV39Fi CAGGAGGGACGATTAATGGC SEQ ID NO: 66 TRAV40FoTGAACTGCACATACACATCC SEQ ID NO: 67 TRAV40Fi ACAGCAAAAACTTCGGAGGC SEQ IDNO: 68 TRAV41Fo AACTGCAGTTACTCGGTAGG SEQ ID NO: 69 TRAV41FiAAGCATGGAAGATTAATTGC SEQ ID NO: 70 TRACRo GCAGACAGACTTGTCACTGG SEQ IDNO: 71 TRACRi AGTCTCTCAGCTGGTACACG SEQ ID NO: 72 TRBV1FoAATGAAACGTGAGCATCTGG SEQ ID NO: 73 TRBV1Fi CATTGAAAACAAGACTGTGC SEQ IDNO: 74 TRBV2Fo GTGTCCCCATCTCTAATCAC SEQ ID NO: 75 TRBV2FiTGAAATCTCAGAGAAGTCTG SEQ ID NO: 76 TRBV3Fo TATGTATTGGTATAAACAGG SEQ IDNO: 77 TRBV3Fi CTCTAAGAAATTTCTGAAGA SEQ ID NO: 78 TRBV4FoGTCTTTGAAATGTGAACAAC SEQ ID NO: 79 TRBV4Fi GGAGCTCATGTTTGTCTACA SEQ IDNO: 80 TRBV5Fo GATCAAAACGAGAGGACAGC SEQ ID NO: 81 TRBV5aFiCAGGGGCCCCAGTTTATCTT SEQ ID NO: 82 TRBV5bFi GAAACARAGGAAACTTCCCT SEQ IDNO: 83 TRBV6aFo GTGTGCCCAGGATATGAACC SEQ ID NO: 84 TRBV6bFoCAGGATATGAGACATAATGC SEQ ID NO: 85 TRBV6aFi GGTATCGACAAGACCCAGGC SEQ IDNO: 86 TRBV6bFi TAGACAAGATCTAGGACTGG SEQ ID NO: 87 TRBV7FoCTCAGGTGTGATCCAATTTC SEQ ID NO: 88 TRBV7aFi TCTAATTTACTTCCAAGGCA SEQ IDNO: 89 TRBV7bFi TCCCAGAGTGATGCTCAACG SEQ ID NO: 90 TRBV7cFiACTTACTTCAATTATGAAGC SEQ ID NO: 91 TRBV7dFi CCAGAATGAAGCTCAACTAG SEQ IDNO: 92 TRBV9Fo GAGACCTCTCTGTGTACTGG SEQ ID NO: 93 TRBV9FiCTCATTCAGTATTATAATGG SEQ ID NO: 94 TRBV10Fo GGAATCACCCAGAGCCCAAG SEQ IDNO: 95 TRBV10Fi GACATGGGCTGAGGCTGATC SEQ ID NO: 96 TRBV11FoCCTAAGGATCGATTTTCTGC SEQ ID NO: 97 TRBV11Fi ACTCTCAAGATCCAGCCTGC SEQ IDNO: 98 TRBV12Fo AGGTGACAGAGATGGGACAA SEQ ID NO: 99 TRBV12aFiTGCAGGGACTGGAATTGCTG SEQ ID NO: 100 TRBV12bFi GTACAGACAGACCATGATGC SEQID NO: 101 TRBV13Fo CTATCCTATCCCTAGACACG SEQ ID NO: 102 TRBV13FiAAGATGCAGAGCGATAAAGG SEQ ID NO: 103 TRBV14Fo AGATGTGACCCAATTTCTGG SEQ IDNO: 104 TRBV14Fi AGTCTAAACAGGATGAGTCC SEQ ID NO: 105 TRBV15FoTCAGACTTTGAACCATAACG SEQ ID NO: 106 TRBV15Fi AAAGATTTTAACAATGAAGC SEQ IDNO: 107 TRBV16Fo TATTGTGCCCCAATAAAAGG SEQ ID NO: 108 TRBV16FiAATGTCTTTGATGAAACAGG SEQ ID NO: 109 TRBV17Fo ATCCATCTTCTGGTCACATG SEQ IDNO: 110 TRBV17Fi AACATTGCAGTTGATTCAGG SEQ ID NO: 111 TRBV18FoGCAGCCCAATGAAAGGACAC SEQ ID NO: 112 TRBV18Fi AATATCATAGATGAGTCAGG SEQ IDNO: 113 TRBV19Fo TGAACAGAATTTGAACCACG SEQ ID NO: 114 TRBV19FiTTTCAGAAAGGAGATATAGC SEQ ID NO: 115 TRBV20Fo TCGAGTGCCGTTCCCTGGAC SEQ IDNO: 116 TRBV20Fi GATGGCAACTTCCAATGAGG SEQ ID NO: 117 TRBV21FoGCAAAGATGGATTGTGTTCC SEQ ID NO: 118 TRBV21Fi CGCTGGAAGAAGAGCTCAAG SEQ IDNO: 119 TRBV23Fo CATTTGGTCAAAGGAAAAGG SEQ ID NO: 120 TRBV23FiGAATGAACAAGTTCTTCAAG SEQ ID NO: 121 TRBV24Fo ATGCTGGAATGTTCTCAGAC SEQ IDNO: 122 TRBV24Fi GTCAAAGATATAAACAAAGG SEQ ID NO: 123 TRBV25FoCTCTGGAATGTTCTCAAACC SEQ ID NO: 124 TRBV25Fi TAATTCCACAGAGAAGGGAG SEQ IDNO: 125 TRBV26Fo CCCAGAATATGAATCATGTT SEQ ID NO: 126 TRBV26FiATTCACCTGGCACTGGGAGC SEQ ID NO: 127 TRBV27Fo TTGTTCTCAGAATATGAACC SEQ IDNO: 128 TRBV27Fi TGAGGTGACTGATAAGGGAG SEQ ID NO: 129 TRBV28FoATGTGTCCAGGATATGGACC SEQ ID NO: 130 TRBV28Fi AAAAGGAGATATTCCTGAGG SEQ IDNO: 131 TRBV29Fo TCACCATGATGTTCTGGTAC SEQ ID NO: 132 TRBV29FiCTGGACAGAGCCTGACACTG SEQ ID NO: 133 TRBV30Fo TGTGGAGGGAACATCAAACC SEQ IDNO: 134 TRBV30Fi TTCTACTCCGTTGGTATTGG SEQ ID NO: 135 TRBCRoGTGTGGCCTTTTGGGTGTGG SEQ ID NO: 136 TRBCRi TCTGATGGCTCAAACACAGC SEQ IDNO: 137 TRDV1Fo TGTATGAAACAAGTTGGTGG SEQ ID NO: 138 TRDV1FiCAGAATGCAAAAAGTGGTCG SEQ ID NO: 139 TRDV2Fo ATGAAAGGAGAAGCGATCGG SEQ IDNO: 140 TRDV2Fi TGGTTTCAAAGACAATTTCC SEQ ID NO: 141 TRDV3FoGACACTGTATATTCAAATCC SEQ ID NO: 142 TRDV3Fi GCAGATTTTACTCAAGGACG SEQ IDNO: 143 TRDCRo AGACAAGCGACATTTGTTCC SEQ ID NO: 144 TRDCRiACGGATGGTTTGGTATGAGG SEQ ID NO: 145 TRGV1-5Fo GGGTCATCTGCTGAAATCAC SEQID NO: 146 TRGV1- AGGAGGGGAAGGCCCCACAG SEQ ID NO: 147 5, 8Fi TRGV8FoGGGTCATCAGCTGTAATCAC SEQ ID NO: 148 TRGV5pFi AGGAGGGGAAGACCCCACAG SEQ IDNO: 149 TRGV9Fo AGCCCGCCTGGAATGTGTGG SEQ ID NO: 150 TRGV9FiGCACTGTCAGAAAGGAATCC SEQ ID NO: 151 TRGV10Fo AAGAAAAGTATTGACATACC SEQ IDNO: 152 TRGV10Fi ATATTGTCTCAACAAAATCC SEQ ID NO: 153 TRGV11FoAGAGTGCCCACATATCTTGG SEQ ID NO: 154 TRGV11Fi GCTCAAGATTGCTCAGGTGG SEQ IDNO: 155 TRGCRo GGATCCCAGAATCGTGTTGC SEQ ID NO: 156 TRGCRiGGTATGTTCCAGCCTTCTGG SEQ ID NO: 157

TABLE 2 Sequence Primer Sequence ID Number IgHV1aFo AGTGAAGGTCTCCTGCAAGGSEQ ID NO: 158 IgHV1bFo AGTGAAGGTTTCCTGCAAGG SEQ ID NO: 159 IgHV1aFiAGTTCCAGGGCAGAGTCAC SEQ ID NO: 160 IgHV1bFi AGTTTCAGGGCAGGGTCAC SEQ IDNO: 161 IgHV1cFi AGTTCCAGGAAAGAGTCAC SEQ ID NO: 162 IgHV1dFiAATTCCAGGACAGAGTCAC SEQ ID NO: 163 IgHV2Fo TCTCTGGGTTCTCACTCAGC SEQ IDNO: 164 IgHV2Fi AAGGCCCTGGAGTGGCTTGC SEQ ID NO: 165 IgHV3aFoTCCCTGAGACTCTCCTGTGC SEQ ID NO: 166 IgHV3bFo CTCTCCTGTGCAGCCTCTGG SEQ IDNO: 167 IgHV3cFo GGTCCCTGAGACTCTCCTGT SEQ ID NO: 168 IgHV3dFoCTGAGACTCTCCTGTGTAGC SEQ ID NO: 169 IgHV3aFi CTCCAGGGAAGGGGCTGG SEQ IDNO: 170 IgHV3bFi GGCTCCAGGCAAGGGGCT SEQ ID NO: 171 IgHV3cFiACTGGGTCCGCCAGGCTCC SEQ ID NO: 172 IgHV3dFi GAAGGGGCTGGAGTGGGT SEQ IDNO: 173 IgHV3eFi AAAAGGTCTGGAGTGGGT SEQ ID NO: 174 IgHV4FoAGACCCTGTCCCTCACCTGC SEQ ID NO: 175 IgHV4Fi AGGGVCTGGAGTGGATTGGG SEQ IDNO: 176 IgHV5Fo GCGCCAGATGCCCGGGAAAG SEQ ID NO: 177 IgHV5FiGGCCASGTCACCATCTCAGC SEQ ID NO: 178 IgHV6Fo CCGGGGACAGTGTCTCTAGC SEQ IDNO: 179 IgHV6Fi GCCTTGAGTGGCTGGGAAGG SEQ ID NO: 180 IgHV7FoGTTTCCTGCAAGGCTTCTGG SEQ ID NO: 181 IgHV7Fi GGCTTGAGTGGATGGGATGG SEQ IDNO: 182 IgHJRo ACCTGAGGAGACGGTGACC SEQ ID NO: 183 IgHJ1RiCAGTGCTGGAAGTATTCAGC SEQ ID NO: 184 IgHJ2Ri AGAGATCGAAGTACCAGTAG SEQ IDNO: 185 IgHJ3Ri CCCCAGATATCAAAAGCATC SEQ ID NO: 186 IgHJ4RiGGCCCCAGTAGTCAAAGTAG SEQ ID NO: 187 IgHJ5Ri CCCAGGGGTCGAACCAGTTG SEQ IDNO: 188 IgHJ6Ri CCCAGACGTCCATGTAGTAG SEQ ID NO: 189 IgKV1FoTAGGAGACAGAGTCACCATC SEQ ID NO: 190 IgKV1Fi TTCAGYGRCAGTGGATCTGG SEQ IDNO: 191 IgKV2Fo GGAGAGCCGGCCTCCATCTC SEQ ID NO: 192 IgKV2aFiTGGTACCTGCAGAAGCCAGG SEQ ID NO: 193 IgKV2bFi CTTCAGCAGAGGCCAGGCCA SEQ IDNO: 194 IgKV3-7Fo GCCTGGTACCAGCAGAAACC SEQ ID NO: 195 IgKV3FiGCCAGGTTCAGTGGCAGTGG SEQ ID NO: 196 IgKV6-7Fi TCGAGGTTCAGTGGCAGTGG SEQID NO: 197 IgKV4-5Fi GACCGATTCAGTGGCAGCGG SEQ ID NO: 198 IgKCRoTTCAACTGCTCATCAGATGG SEQ ID NO: 199 IgKCRi ATGAAGACAGATGGTGCAGC SEQ IDNO: 200 IgLV1aFo GGGCAGAGGGTCACCATCTC SEQ ID NO: 201 IgLV1bFoGGACAGAAGGTCACCATCTC SEQ ID NO: 202 IgLV1aFi TGGTACCAGCAGCTCCCAGG SEQ IDNO: 203 IgLV1bFi TGGTACCAGCAGCTTCCAGG SEQ ID NO: 204 IgLV2FoCTGCACTGGAACCAGCAGTG SEQ ID NO: 205 IgLV2Fi TCTCTGGCTCCAAGTCTGGC SEQ IDNO: 206 IgLV3aFo ACCAGCAGAAGCCAGGCCAG SEQ ID NO: 207 IgLV3bFoGAAGCCAGGACAGGCCCCTG SEQ ID NO: 208 IgLV3aFi CTGAGCGATTCTCTGGCTCC SEQ IDNO: 209 IgLV3bFi TTCTCTGGGTCCACCTCAGG SEQ ID NO: 210 IgLV3cFiTTCTCTGGCTCCAGCTCAGG SEQ ID NO: 211 IgLV4Fo TCGGTCAAGCTCACCTGCAC SEQ IDNO: 212 IgLV4Fi GGGCTGACCGCTACCTCACC SEQ ID NO: 213 IgLV5FoCAGCCTGTGCTGACTCAGCC SEQ ID NO: 214 IgLV5Fi CCAGCCGCTTCTCTGGATCC SEQ IDNO: 215 IgLV6Fo CCATCTCCTGCACCCGCAGC SEQ ID NO: 216 IgLV7-8FoTCCCCWGGAGGGACAGTCAC SEQ ID NO: 217 IgLV9, CTCMCCTGCACCCTGAGCAG SEQ IDNO: 218 11Fo IgLV10Fo AGACCGCCACACTCACCTGC SEQ ID NO: 219 IgLV6, 8FiCTGATCGSTTCTCTGGCTCC SEQ ID NO: 220 IgLV7Fi CTGCCCGGTTCTCAGGCTCC SEQ IDNO: 221 IgLV9Fi ATCCAGGAAGAGGATGAGAG SEQ ID NO: 222 IgLV10-CTCCAGCCTGAGGACGAGGC SEQ ID NO: 223 11Fi IgLC1-7Ro GCTCCCGGGTAGAAGTCACTSEQ ID NO: 224 IgLC1-7Ri AGTGTGGCCTTGTTGGCTTG SEQ ID NO: 225

TABLE 3 Primer Sequence Sequence ID Number HLAI CCCACTCCATGAGGTATTTC SEQID NO: 226 Fo11 HLAI CCTACTCCATGAGGTATTTC SEQ ID NO: 227 Fo12 HLAIGCGGGGAGCCCCGCTTCATC SEQ ID NO: 228 Fo31 HLAI GCGGGAAGCCCCGCTTCATC SEQID NO: 229 Fo32 HLAI GTGGAGAGCCCCGCTTCATC SEQ ID NO: 230 Fo33 HLAIGCGGAAAGCCCCGCTTCATC SEQ ID NO: 231 Fo34 HLAI GCGGAAAGCCCCACTTCATC SEQID NO: 232 Fo35 HLAI GCGGGAAGCCCCACTTCATC SEQ ID NO: 233 Fo36 HLAIGTGGGCTACGTGGACGACAC SEQ ID NO: 234 Fi11 HLAI GTGGGCTACGTGGACGGCAC SEQID NO: 235 Fi12 HLAI- GTTCGTGCGGTTCGACAGCG SEQ ID NO: 236 Fi21 HLAI-GTTCGTGCGGTTTGACAGCG SEQ ID NO: 237 Fi22 HLAI- GTTCGTGAGGTTCGACAGCG SEQID NO: 238 Fi23 HLAI TAATCCTTGCCGTCGTAGGC SEQ ID NO: 239 Ri11 HLAITAATCCTTGCCGTCGTAAGC SEQ ID NO: 240 Ri12 HLAI TAATCTTTGCCGTCGTAGGC SEQID NO: 241 Ri13 HLAI GGTCCTCGTTCAGGGCGATG SEQ ID NO: 242 Ro11 HLAIGGTCCTCTTTCAGGGCGATG SEQ ID NO: 243 Ro12 HLAI GGTCCTCGTTCAAGGCGATG SEQID NO: 244 Ro13 HLAI GATCCTCGTTCAGGGCGATG SEQ ID NO: 245 Ro14 HLAIGGTCCTCATTCAGGGCGATG SEQ ID NO: 246 Ro15 HLAI- GCCTACGACGGCAAGGATTA SEQID NO: 247 Fo41 HLAI- GCTTACGACGGCAAGGATTA SEQ ID NO: 248 Fo42 HLAI-GCCTACGACGGCAAAGATTA SEQ ID NO: 249 Fo43 HLAI- CATCGCCCTGAACGAGGACC SEQID NO: 250 Fi31 HLAI- CATCGCCCTGAAAGAGGACC SEQ ID NO: 251 Fi32 HLAI-CATCGCCTTGAACGAGGACC SEQ ID NO: 252 Fi33 HLAI- CATCGCCCTGAACGAGGATC SEQID NO: 253 Fi34 HLAI- CATCGCCCTGAATGAGGACC SEQ ID NO: 254 Fi35 HLAI-GGTATCTGCGGAGCCCGTCC SEQ ID NO: 255 Ri21 HLAI- GGTATCTGCGGAGCCACTCC SEQID NO: 256 Ri22 HLAI- GGTGTCTGCGGAGCCACTCC SEQ ID NO: 257 Ri23 HLAI-GGTATCCGCGGAGCCACTCC SEQ ID NO: 258 Ri24 HLAI- GCAGCGTCTCCTTCCCGTTC SEQID NO: 259 Ro21 HLAI- CCAGCTTGTCCTTCCCGTTC SEQ ID NO: 260 Ro22 HLAI-CCAGCGTGTCCTTCCCGTTC SEQ ID NO: 261 Ro23 HLAI- GCAGCGTCTCCTTCCCATTC SEQID NO: 262 Ro24 HLAI- GCAGCGTCTCCTTCCKGTTC SEQ ID NO: 263 Ro25 DRB17TGTCATTTCTTCAATGGGAC SEQ ID NO: 264 Fo11 DRB1 AGTGTCATTTCTTCAACGGG SEQID NO: 265 Fo12 DRB1 GTGTTATTTCTTCAATGGGA SEQ ID NO: 266 Fo13 DRB1GTGTCAATTCTTCAATGGGA SEQ ID NO: 267 Fo14 DRB4 Fo GTGTCATTTCCTCAATGGGASEQ ID NO: 268 DRB1 GGAGCGGGTGCGGTTGCTGG SEQ ID NO: 269 Fi11 DRB1GGAGCGGGTGCGGTACCTGG SEQ ID NO: 270 Fi12 DRB1 GGAGCGGGTGCGGTTCCTGG SEQID NO: 271 Fi13 DRB1 GGAGCGGGTGCGATTCCTGG SEQ ID NO: 272 Fi14 DRB1GGAGCGGGTGCGGTATCTGC SEQ ID NO: 273 Fi15 DRB1 GGAGCGGGTGCGGTTACTGG SEQID NO: 274 Fi16 DRB45 Fi ACATCTATAACCAAGAGGAG SEQ ID NO: 275 DRB6 FiACATCCATAAACGGGAGGAG SEQ ID NO: 276 DRB7 Fi TATAACCAAGAGGAGTACGT SEQ IDNO: 277 DRB Ri11 AACCCCGTAGTTGTGTCTGC SEQ ID NO: 278 DRB4 RiAACCCCGTAGTTGTATCTGC SEQ ID NO: 279 DRB6 Ri CGTAATTGTATCTGCAGTAG SEQ IDNO: 280 DRB7 Ri TAGTTGTCCACTTCGGCCCG SEQ ID NO: 281 DRB Ro1CGCTGCACTGTGAAGCTCTC SEQ ID NO: 282 DRB Ro2 CGCTGCACCGTGAAGCTCTC SEQ IDNO: 283 DRA Fo CCTGTGGAACTGAGAGAGCC SEQ ID NO: 284 DRA FiCAACGTCCTCATCTGTTTCA SEQ ID NO: 285 DRA Ri CTGCTGCATTGCTTTTGCGC SEQ IDNO: 286 DRA Ro TTACAGAGGCCCCCTGCGTT SEQ ID NO: 287 DPB FoGTCCAGGGCAGGGCCACTCC SEQ ID NO: 288 DPB Fi11 AATTACGTGTACCAGGGACG SEQ IDNO: 289 DPB Fi12 AATTACCTTTTCCAGGGACG SEQ ID NO: 290 DPB Fi13AATTACGTGTACCAGTTACG SEQ ID NO: 291 DPB Fi14 AATTACGCGTACCAGTTACG SEQ IDNO: 292 DPB Ri11 CGGCCTCGTCCAGCTCGTAG SEQ ID NO: 293 DPB Ri12TGGGCCCGCCCAGCTCGTAG SEQ ID NO: 294 DPB Ri13 TGGGCCCGACCAGCTCGTAG SEQ IDNO: 295 DPB Ro GGACTCGGCGCTGCAGGGTC SEQ ID NO: 296 DPA FoAGGAGCTGGGGCCATCAAGG SEQ ID NO: 297 DPA Fi GACCATGTGTCAACTTATGC SEQ IDNO: 298 DPA Ri1 CTCAGGGGGATCGTTGGTGG SEQ ID NO: 299 DPA Ri2CTCAGGGGGATCATTGGCGG SEQ ID NO: 300 DPA Ro CAGCTCCACAGGCTCCTTGG SEQ IDNO: 301 DQB Fo ACTCTCCCGAGGATTTCGTG SEQ ID NO: 302 DQB FiTGTGCTACTTCACCAACGGG SEQ ID NO: 303 DQB Ri1 ACCTCGTAGTTGTGTCTGCA SEQ IDNO: 304 DQB Ri2 AACTGGTAGTTGTGTCTGCA SEQ ID NO: 305 DQB Ro1ACTCTCCTCTGCAGGATCCC SEQ ID NO: 306 DQB Ro2 ACTCGCCGCTGCAAGGTCGT SEQ IDNO: 307 DQB Ro3 ACTCTCCTCTGCAAGATCCC SEQ ID NO: 308 DQA FoTGGTGTAAACTTGTACCAGT SEQ ID NO: 309 DQA Fi ACCCATGAATTTGATGGAGA SEQ IDNO: 310 DQA Ri GGAACCTCATTGGTAGCAGC SEQ ID NO: 311 DQA RoACTTGGAAAACACTGTGACC SEQ ID NO: 312

1. A method for producing an immune status profile for a human and/oranimal, the method comprising (a) amplifying, in a first amplificationusing target-specific primers, at least one DNA and/or RNA from a sampleof white blood cells from a human or animal subject to produce at leastone first amplicon, at least a portion of the target-specific primerscomprising additional nucleotides to incorporate into the at least onefirst amplicon a binding site for a common primer; (b) rescuing the atleast one first amplicon from the first amplification; (c) amplifying,in a second amplification using at least one common primer, the at leastone first amplicon to produce at least one second amplicon; and (d)sequencing the at least one second amplicon to identify and quantify oneor more DNA sequences representing rearrangements to create an immunestatus profile.
 2. The method of claim 1 further comprising the steps of(e) inputting the one or more DNA sequences into a database to providedata which may be stored on a computer, server, or other electronicstorage device, (f) inputting a data set of identifying information andcharacteristics for an individual corresponding to the sequences as datawhich may be stored on a computer, server, or other electronic storagedevice, and (g) evaluating the data of step (e) and step (f) for one ormore individuals to determine whether a correlation exists between theone or more sequences and one or more characteristics of the individualcorresponding to the one or more sequences.
 3. The method of claim 1wherein the target-specific primers are chosen from among the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57,SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ IDNO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76,SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO:81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ IDNO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQID NO: 137, SEQ ID NO:138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO:151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO:160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO:169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO:178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO:187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO:196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO:205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO:214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO:223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO:232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO:241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO:250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO:259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO:268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO:277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO:286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO:295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO:304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, andcombinations thereof.
 4. A method for producing an immune responseprofile, the method comprising: amplifying RNA and/or DNA from a humanor animal subject using target-specific primers chosen from among thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57,SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ IDNO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76,SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO:81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ IDNO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQID NO: 137, SEQ ID NO:138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO:151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO:160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO:169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO:178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO:187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO:196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO:205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO:214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO:223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO:232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO:241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO:250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO:259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO:268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO:277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO:286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO:295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO:304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, andcombinations thereof to perform a first amplification producing at leastone first amplicon, at least one of the target-specific primerscontaining additional base pairs so that the amplification results inthe addition of a binding sequence for at least one common primer intothe at least one first amplicon; amplifying the at least one firstamplicon in a second amplification using at least one common primer toproduce at least one second amplicon; and sequencing the at least onesecond amplicon to identify and quantify the sequences produced by thefirst and second amplifications.